Project Summary While there have been advances in targeting actionable oncogenes such as ALK fusion proteins, the majority of non-small cell lung cancer patients feature either difficult to target mutations, e.g. KRAS, or lack known oncogenic drivers. These patients do not derive much benefit from currently approved therapies. Therefore, in order to find effective treatments for these patients, novel strategies need to be implemented to find therapies that are able to kill lung cancer cells and can be rapidly implemented clinically. Many targeted drugs show potent anticancer activity in subsets of NSCLC, which is often unrelated to inhibition of the cognate targets of these drugs. These beneficial off-targets can be exploited to identify novel drug repurposing opportunities that allow for the treatment of these difficult to treat cancers. Detailed drug profiling is best done using unbiased chemical and systems biology approaches. Chemical proteomic experiments investigate direct drug-protein interactomes giving a number of possible candidate targets. These interactomes can be integrated with global signaling changes measured by phosphoproteomics to give a comprehensive view of the mechanism of action of a drug. Ceritinib is an FDA-approved ALK/IGF1R inhibitor for the treatment of ALK positive non-small cell lung cancer (NSCLC) and we observed that ceritinib has unexpected activity in KRAS mutant NSCLC. A focused chemical proteomic profiling of ceritinib has identified a number of novel targets of ceritinib including FAK1 and RSK1/2. Preliminary studies into ceritinib?s mechanism of action have shown alterations in the cross talk signaling between the KRAS and RHOA pathways. However, the functional relevance and exact molecular mechanisms of such regulation remain unclear. Finally, based on this mechanism we designed a novel drug combination strategy combining ceritinib with the FDA-approved tubulin inhibitor, paclitaxel. We will test the hypothesis that the molecular determinants following inhibition of FAK1 and RSK1/2 by ceritinib carry predictive ability for ceritinib and paclitaxel combination efficacy by achieving the following aims. Aim 2.1: To evaluate the role of YB1 in the observed synergy between ceritinib and paclitaxel. Aim 2.2: To investigate the role of GEF-H1 in ceritinib?s mechanism of action. Aim 2.3: To characterize both ceritinib monotherapy and paclitaxel combination therapy in KRAS-mutant NSCLC PDX models. We will then have a greater understanding of the relevant downstream molecular determinants of ceritinib?s mechanism of action and of the observed synergy with paclitaxel. This will ultimately lead to predictive capabilities for ceritinib sensitivity and further clinical development of ceritinib to treat patients whom will benefit.